Sustained low micromolar hydrogen peroxide exposure induces sequential red blood cell dysfunction

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Introduction: storage. Most experimental oxidative stress models rely on supraphysiological bolus oxidant addition, yet this approach does not replicate the sustained, low-level oxidant exposure encountered physiologically. Understanding the temporal sequence of RBC oxidative injury under physiologically relevant conditions is essential for identifying early biomarkers of dysfunction and developing targeted interventions. Methods: ) at low micromolar concentrations (0.8-8 µM) and examined the temporal progression of oxidative damage in human RBCs over 24 hours. Healthy donor RBCs exposed to GX (1-10 mU/mL) were evaluated for oxidative burden, antioxidant status, hemoglobin oxidation, relative hemoglobin release, membrane integrity/viability by calcein fluorescence, deformability by ektacytometry, phosphatidylserine (PS) externalization by Annexin V binding, and vesiculation by microscopy. Results: exposure induced rapid glutathione depletion (within 6 hours) followed by progressive methemoglobin formation. Single-cell analyses demonstrated a strong inverse relationship between intracellular oxidative autofluorescence and cellular dysfunction. GX induced concentration-dependent impairment of RBC deformability, with 10 mU/mL causing significant membrane rigidity and hemoglobin release indicative of membrane lysis by 24 hours. Time-dependent vesiculation and release of CD235a- and Band 3-labeled microvesicles occurred with vesiculating RBCs exhibiting higher oxidative burden than non-vesiculating cells. Notably, PS externalization was absent on both vesiculating RBCs and their shed microvesicles. Discussion: These findings define a temporal hierarchy of oxidative injury under physiologically relevant conditions and demonstrate that vesiculation and PS externalization are mechanistically uncoupled under sustained oxidative stress. This model provides a framework for identifying early biomarkers of RBC dysfunction that may guide the development of targeted interventions to optimize blood storage and mitigate oxidative injury.

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